P604: catalase expression in leukemia cells is controlled by genetic and epigenetic mechanisms

Background: Chronic lymphocytic leukemia (CLL) is the most prevalent form of leukemia in Western countries. It is an incurable disease characterized by an extremely variable clinical course and response to treatment. We have recently shown that high catalase (CAT) expression identifies a more aggressive clinical behavior in CLL. However, molecular mechanisms controlling catalase expression in leukemia cells are still poorly characterized. The rs1001179 single nucleotide polymorphism (SNP) as well as DNA methylation in the CAT promoter have been shown to regulate CAT expression in various cell types. Aims: With the aim to characterize regulatory mechanisms underlying differential expression of catalase in CLL, in this study we investigated the role of rs1001179 SNP and CpG Island II methylation encompassing this SNP in the regulation of CAT expression. Methods: Peripheral blood mononuclear cells (PBMC) from 55 CLL patients and 50 healthy donors (HD) were used for the study. CAT mRNA levels were measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR). DNA samples were genotyped for the rs1001179 SNP using Restriction Fragment Length Polymorphism (RFLP)-PCR. Bioinformatic analysis was used to predict transcription factor (TF) binding to SNP sequences. Chromatin Immunoprecipitation (ChIP)-qPCR was performed to validate predicted TF binding sites. Methylation levels of CpG sites within the CAT promoter was determined by pyrosequencing of bisulfite-converted DNA. Results: The rs1001179 SNP genotyping showed that CLL cells harboring the T allele exhibited a significantly higher catalase expression compared with cells bearing the CC genotype. Interestingly, bioinformatic analysis predicted that the SNP provide distinct binding sites for various TF, including ETS-1 and GR-β. ChIP assay confirmed that CAT promoter harboring the T -but not C- allele was accessible to ETS-1 and GR-β, but not to the other analyzed TFs. In addition, CLL cells exhibited lower methylation levels within the CAT promoter compared with HD B cells, in line with the higher catalase mRNA and protein levels expressed by CLL versus HD B cells. Moreover, methylation levels negatively correlated with CAT expression in CLL cells. Remarkably, inhibition of methyltransferase activity in leukemic cells induced a significant increase of CAT mRNA and protein levels, thus showing that DNA methylation could control catalase expression in CLL. Moreover, expression of DNA methyl transferase 1 (DNMT1) resulted significantly reduced in CLL cells compared with HD B cells and inversely correlated with CAT expression in CLL, thus suggesting that differences in methylation levels underlying catalase expression could be driven by the DNMT1 enzyme. Finally, modeling analysis showed that the CT/TT genotypes exhibited a lower methylation and a higher CAT expression level, suggesting that the rs1001179 T allele and methylation cooperate in inducing CAT gene expression. Summary/Conclusion: Our data show that SNP as well as methylation of the catalase promoter are involved in controlling catalase expression in CLL. The key advance of this study is to provide new insights into the regulatory mechanisms underlying differential expression of catalase in leukemic cells, which is of clinical relevance in CLL. Moreover, our study may form the basis for future challenges aimed at developing combinatorial therapies targeting catalase regulatory pathways.